Sorting motifs in the cytoplasmic tail of the immunomodulatory E3/49K protein of species D adenoviruses modulate cell surface expression and ectodomain shedding.

The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved resulting in secretion of its large ectodomain (sec49K). Sec49K binds to CD45 on leukocytes, impairing activation and functions of NK cells and T cells. E3/49K is localized in the Golgi/trans-Golgi-network (TGN), early endosomes and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly-synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YxxΦ and LL that are important for binding the clathrin adaptor proteins AP-1 and AP-2 in vitro. Surprisingly, mutating the LL motif, either alone or together with YxxΦ, did not prevent proteolytic processing, but increased cell surface expression and secretion. Upon Brefeldin-A treatment cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix-metalloproteases.